Protein microarrays provide a powerful tool for the study of protein function and protein-protein interactions. In particular, protein microarrays have been used to investigate protein interaction with various drugs, antibodies, lipids, nucleic acids, and other proteins. Protein microarrays currently are available in two general formats: antibody arrays and target protein arrays. Antibody arrays contain an array of antibodies that measure the abundance of specific proteins in samples (see, e.g., Haab et al., Genome Biology, 2: research004.1-0004.13 (2001). Target protein arrays, on the other hand, contain an array of proteins of interest that are used to measure the abundance of proteins in response to specific exogenous stimuli (e.g., drugs, antibodies, lipids, etc.), or to identify enzyme substrates (see, e.g., Cahill et al., Adv. Biochem. Eng. Biotechnol., 83: 177 (2003) and Jona et al., Curr. Opin. Mol. Ther., 5: 271 (2003)).
Target protein microarrays typically are generated in two steps. First, proteins are separately produced, and then applied (or “spotted”) on the array surface using a variety of linkage chemistries (see, e.g., Lueking et al., Anal. Biochem., 270: 103 (1999), MacBeath et al., Science, 289: 1760 (2000), Zhu et al., Science, 293: 2101 (2001), and Newman et al., Science, 300: 2097 (2003)). Despite their demonstrated utility, the widespread use of target protein microarrays has been limited by a number of factors. For example, current protein microarray technologies are labor-intensive. In addition, currently there are no high-throughput expression systems that produce significant yields of mammalian proteins of sufficiently high purity. Moreover, protein instability, both before and after spotting on the array, is another obstacle to the implementation of target protein microarrays on a large-scale.
To circumvent the problems associated with current protein microarray technology, researchers have developed new systems in which immobilized DNA molecules are transcribed and translated on the microarray in situ, whereupon newly synthesized proteins are immobilized on the microarray surface at the site of expression. For example, Nord et al., J Biotech., 106: 1-13 (2003) discloses an array technology called microbead display of proteins. In this technology, proteins are captured by antigen-antibody binding as they are synthesized. Specifically, biotin labeled PCR products (containing a bacteriophage T7 promoter and a FLAG epitope in-frame with two IgG binding domains) are first anchored onto microbeads through streptavidin-biotin affinity binding. Anti-FLAG antibody also is immobilized onto the same microbead. The beads are then incubated with a coupled cell-free transcription-translation extract to produce the corresponding protein. The newly synthesized proteins are trapped via FLAG peptide-FLAG antibody interaction. In addition, Ramachandran et al., Science, 305: 86-90 (2004), discloses a similar antibody-mediated protein microarray format. In this case, purified plasmids are arrayed on a microscope slide through biotin-avidin binding. The genes encoded by the plasmids are fused with glutathione-S-transferase (GST) protein to produce GST-fusion proteins. The slides also are printed with polyclonal GST antibody to capture the newly synthesized GST-fusion proteins following coupled cell-free transcription-translation reactions. Other methods for generating protein microarrays utilizing direct immobilization of proteins synthesized in situ are disclosed in, for example, He, Methods in Molecular Biology, 264: 25-31 (2004) and International Patent Application Publication WO 02/14860.
While the above methods have met with some success, their widespread use is limited by a number of factors. First, both methods require a second protein, i.e., an antibody, to capture the synthesized protein of interest. Antibody generation and purification adds time and cost to the process. Second, methods for maintaining long-term antibody stability, and therefore, array stability, have yet to be developed.
Accordingly, there remains a need for more robust and stable protein microarrays and more efficient methods for producing such protein microarrays. The invention provides such microarrays and methods. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.